Human IL-23 is essential for IFN-γ-dependent immunity to mycobacteria

Patients with autosomal recessive (AR) IL-12p40 or IL-12Rβ1 deficiency display Mendelian susceptibility to mycobacterial disease (MSMD) due to impaired IFN-γ production and, less commonly, chronic mucocutaneous candidiasis (CMC) due to impaired IL-17A/F production. We report six patients from four kindreds with AR IL-23R deficiency. These patients are homozygous for one of four different loss-of-function IL23R variants. All six patients have a history of MSMD but only two suffered from CMC. We show that IL-23 induces IL-17A only in MAIT cells, possibly contributing to the incomplete penetrance of CMC in patients unresponsive to IL-23. By contrast, IL-23 is required for both baseline and Mycobacterium-inducible IFN-γ immunity in both Vδ2+ γδ T and MAIT cells, probably contributing to the higher penetrance of MSMD in these patients. Human IL-23 appears to contribute to IL-17A/F-dependent immunity to Candida in a single lymphocyte subset, but is required for IFN-γ-dependent immunity to Mycobacterium in at least two lymphocyte subsets.


INTRODUCTION
Life-threatening disease during primary infection in otherwise healthy individuals can result from monogenic inborn errors of immunity (IEI) (1,2). Studies of such IEIs can shed light on the essential and redundant roles of the corresponding human genes in host defense in natura, while clarifying mechanisms of disease (3)(4)(5)(6). Mendelian susceptibility to This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
whereas IL-23 is a signature inducer of IL-17A/F (49)(50)(51)(52). We investigated the role of human IL-23 in host defense further, by searching for new patients with AR IL-23R deficiency in a cohort of >15,000 patients with diverse infectious diseases.

Six patients from four kindreds homozygous for private IL23R variants
We searched for non-synonymous, essential splice site, or copy number variants of IL23R in a cohort of >15,000 patients with highly diverse infectious diseases, including 1,618 patients with MSMD, 1,454 with TB, and 730 with CMC. The cohorts overlap due to the occurrence of syndromic forms of infection. We selected rare variants (MAF<0.01) that were homozygous or potentially compound heterozygous. Six patients from four kindreds were homozygous for private IL23R variants (NM_144701.2) ( Figure 1A). P1 and P2 from Kindred A, reported in previous studies (11,22), are homozygous for the c.344G>A missense variant, which results in the p.C115Y substitution in a key domain of IL-23R ( Figure 1B) (11,53). P3, from Kindred B, is homozygous for the c.367+1G>A variant, which is predicted to alter the canonical donor splice site of exon 3 ( Figure  1A and 1B). P4 from Kindred C, is homozygous for the c.1149-1G>A variant, which is predicted to alter the canonical acceptor splice site of exon 10 ( Figure 1A and 1B). Finally, P5 and P6, from Kindred D, are homozygous for the nonsense c.805G>T variant (p.E269*) upstream from the segment encoding the transmembrane domain of IL-23R ( Figure 1A and 1B) (53). All heterozygous or homozygous wild-type (WT) relatives of these patients were healthy ( Figure 1A). The four IL23R variants found in kindreds A-D are not reported in any individuals of the Genome Aggregation Database (gnomAD) v2.1.1 (https://gnomad.broadinstitute.org/), ATAVDB (http://atavdb.org), Great Middle East Database (http://igm.ucsd.edu/gme), the Iranome database (http://www.iranome.com/), or in any other individuals from our in-house database (>15,000 individuals). Finally, these four IL23R variants are proven (p.C115Y) or predicted to be loss-of-function (pLOF) (c.367+1G>A, c.1149-1G>A, p.E269*), with combined annotation-dependent depletion (CADD) scores well above the mutation significance cutoff (MSC) of IL23R ( Figure 1C) (54)(55)(56). These data suggest that these six patients have AR IL-23R deficiency.

Six patients with MSMD, including two with CMC
The six patients belonged to four unrelated families originating from and living in Iran. Principal component analysis on whole-exome sequencing (WES) data confirmed the Iranian ancestry of P5 and P6, whereas P1 to P4 were closer to the European population ( Figure S1A) (57). The high rate of homozygosity in these patients was suggestive of familial consanguinity ( Figure S1B) (58), and their kinship coefficients suggested that the four kindreds were not related to each other (59). P1 (Kindred A), born in 1994 to consanguineous parents, was vaccinated with BCG in early infancy and developed BCGadenitis (BCG-itis), which persisted for one year before spontaneously resolving (Table S1). Her brother, P2, born in 2006, was also vaccinated with BCG early in infancy, leading to BCG-itis, which progressed to disseminated BCG disease (BCG-osis) despite antibiotic treatment, resulting in the death of this patient at the age of eight years. P3 (Kindred B), born in 2010 to consanguineous parents, was vaccinated with BCG and developed BCG-osis (22). P4 (Kindred C), born in 2008 to consanguineous parents, was vaccinated with BCG and developed BCG-itis, which persisted for three years before spontaneously resolving. P4 has also suffered from oral and esophageal candidiasis, which persisted between the ages of three months and ten years. He was treated with fluconazole, and no recurrence has been observed on prophylaxis. P5 (Kindred D), born in 2015 to consanguineous parents, was vaccinated with BCG and developed axillary BCG adenitis (BCG-itis) at eight months, with spontaneous resolution at 12 months. His brother P6, born in 2019, was vaccinated with BCG and developed recurrent axillary BCG-itis, which resolved at 12 months after abscess drainage and antimycobacterial therapy until the age of 18 months. P6 has also presented one episode of oral candidiasis, which resolved on fluconazole. No infections with other intramacrophagic pathogens, such as environmental mycobacteria and Salmonella, were observed in any of the six patients, despite such infections being common in patients with IL-12Rβ1 and IL-12p40 deficiencies. Clinical penetrance was complete for BCG disease, but incomplete for CMC (two of the six patients) (Table S1). All six patients have been infected with diverse viruses and bacteria ( Figure S1C), without severe clinical consequences. WES analysis of the six patients found no other homozygous candidate variants of known MSMD-or CMC-causing genes. Other rare homozygous or hemizygous coding and splice-site (including essential splice site) variants were present, with CADD scores above the corresponding MSC scores, and these variants were not present in the homozygous or hemizygous state in GnomAD v2.1.1. However, none of these variants were related to IFN-γ or IL-17 immunity ( Figure S1B and Table S2). Overall, the six patients with a putative diagnosis of AR IL-23R deficiency had an MSMD phenotype, and two also had CMC.

Population genetics of the IL23R locus
No IL23R pLOF variants were found in the homozygous state in the general population.
Only 13 in-frame variants were found in the homozygous state; all were missense variants with MAFs ranging from 5.3 × 10 −5 to 8.8 × 10 −1 ( Figure 1C). We reported all of these variants to be neutral in an overexpression assay in a previous study (11). A revised analysis of the population genetics of IL23R showed that the situation had not changed since 2018. The IL23R locus is subject to negative selection, with a consensus-based measure of negative selection (CoNeS) score of −0.08, consistent with an AR IEI ( Figure 1D) (60), and an intermediate gene damage index (GDI) of 3.6 (61). Similarly, we found no pLOF variants and only one in-frame variant of IL23A in the homozygous state in the general population. Overall, we found six MSMD patients from four kindreds homozygous for an experimentally proven missense LOF (eLOF) (kindred A) or pLOF (kindreds B, C, D) variant. A significant enrichment in homozygosity for such variants, which were found exclusively in patients with MSMD, was observed in the 802 MSMD patients with no identified genetic etiology relative to >3,000 patients with diverse infectious diseases from our database and the general population (p=5.7 × 10 −4 and p=3.5 × 10 −9 , respectively).
The absence of homozygous pLOF variants in the TB cohort, which includes patients without MSMD from countries in which BCG vaccination is mandatory, further suggests that AR IL-23R deficiency displays complete penetrance for BCG disease. Collectively, these findings strongly suggest that the six patients have MSMD (and CMC, for two of the patients) because of AR IL-23 deficiency.

Two mutant alleles of IL23R disrupt mRNA splicing
The c.367+1G>A and c.1149-1G>A IL23R variants affect essential splice sites. We therefore hypothesized that they would produce aberrantly spliced transcripts. We first tested their effect on IL23R mRNA splicing by performing exon trapping. Both c.367+1G>A and c.1149-1G>A IL23R mutant mRNAs were aberrant, as shown by comparison with cells transfected with wild-type (WT) IL23R ( Figure S1D and S1E). We then used mRNA extracted from Epstein-Barr virus-immortalized B lymphocytes (EBV-B cells) from a healthy control and P3, and from activated and expanded T cells (T-blasts) from a healthy control and P4, to generate and amplify IL23R cDNAs from exons 2 to 5 (healthy control and P3) or exons 8 to 11 (healthy control and P4). Sanger sequencing of the PCR products followed by TOPO-TA cloning showed a match to the canonical transcript for >90% of cDNAs from the mRNA isolated from healthy control cells, but none of those from the mRNA from patient cells ( Figure S1F and S1G). Three transcripts were detected in P3's EBV-B cells. Two of these transcripts predominated and were predicted to encode a protein with an in-frame partial deletion of the extracellular domain of IL-23R (G45_Y123delinsD, G24_Y123delinsD). The minor transcript was predicted to encode a truncated protein (G45Ffs*16) ( Figure 1E). All four transcripts observed in P4 T-blasts were predicted to encode truncated proteins ( Figure 1E). Thus, c.367+1 G>A and c.1149-1G>A led to aberrantly spliced and pLOF IL23R mRNAs in P3 and P4, respectively, at least in the cell types tested, with no leakiness. These findings suggest that these patients had AR IL-23R deficiency.

Four loss-of-function IL23R alleles
We then investigated the expression and function of the four mutant alleles. As a surrogate for these alleles, we generated cDNAs corresponding to the variants of P1/P2 (C115Y), and P5/P6 (E269*) and to the transcripts observed in the cells of P3 (G45_Y123delinsD, G24_Y123delinsD and G45Ffs*16) and P4 (I384Kfs*6, I384Lfs*8, D349Gfs*3, and N350*). We used lentivirus-mediated gene transfer to ensure the stable expression of WT or mutant IL23R cDNA in an EBV-B cell line that endogenously expressed STAT3 and IL-12Rβ1, but not IL-23R, and had been engineered to express STAT4 in a stable manner (referred to here as EBV-B STAT4 ) (11,62). Transduction efficiency was assessed by measuring the cell-surface expression of ΔNGFR (CD271, incorporated into the backbone of the lentiviral plasmid) ( Figure S2A) (63). EBV-B STAT4 cells transduced with the WT or mutant IL23R cDNAs produced similar amounts of IL23R mRNA ( Figure S2B). However, relative to EBV-B STAT4 cells transduced with WT IL23R cDNA, EBV-B STAT4 cells transduced with mutant IL23R cDNA had similar (G24_Y123delinsD or G45_Y123delinsD), weaker (C115Y, I384Kfs*6, I384Lfs*8), or no (G45Ffs*16, N349Gfs*3, N350*, E269*) cell-surface expression of IL-23R (Figure 2A and S2C). We then stimulated EBV-B STAT4 cells expressing WT or mutant IL-23R proteins with IL-23, or IFN-α2b as a control. Human IL-23-dependent signaling via IL-12Rβ1/IL-23R in these cells preferentially results in STAT3 activation. EBV-B STAT4 cells expressing WT, but not mutant IL-23R phosphorylated STAT3 in response to IL-23, whereas all cells phosphorylated STAT3 in response to IFN-α2b ( Figure 2B and S2D). All the IL23R mutant alleles were, therefore, LOF for responses to IL-23. These findings strongly suggest that the patients had AR complete IL-23R deficiency.

Six patients with AR complete IL-23R deficiency
Our next set of experiments used patient-derived cells to capture the effects of the patients' full genotypes at these loci in the context of their own genome. We assessed the cell-surface expression and function of IL-23R in EBV-B cells from P1 and P3. Relative to EBV-B cells from a healthy control or an IL-12Rβ1-deficient patient, cell-surface IL-23R expression was normal in P1 and much weaker in P3 ( Figure S2E). These EBV-B cells were then left unstimulated or stimulated with IL-23 or IFN-α2b. EBV-B cells from the healthy control responded to IL-23 by phosphorylating STAT3, whereas EBV-B cells from P1, P3, and an IL-12Rβ1-deficient patient did not. By contrast, all cells phosphorylated STAT3 in response to IFN-α2b ( Figure 2C). We then assessed the function of IL-23R in T-blasts. T-blasts from healthy controls (including P4's sister, who is WT for IL-23R), P4, P5, and P6, and an IL-12Rβ1-deficient patient were left unstimulated, or were stimulated with IL-23 or IFN-α2b. T-blasts from healthy controls responded to IL-23 by phosphorylating STAT3, whereas T-blasts from P4, P5, P6, and the IL12Rβ1-deficient patient did not ( Figure 2D and Figure S2F). T-blasts from healthy controls, P4, P5, P6, and an IL-12Rβ1 deficient patient responded to IFN-α2b by phosphorylating STAT3 or STAT4, and to IL-12 by phosphorylating STAT4, with the exception of IL-12Rβ1 deficient T-blasts ( Figure 2D, and Figure S2F and S2G). Finally, whole blood from two healthy individuals, including a travel control, P5 and P6, was left unstimulated, or stimulated with IL-23 or IFN-α2b, and STAT phosphorylation was assessed by mass cytometry by time-of-flight (CyTOF) ( Figure  2E and Figure S2H). In cells from healthy donors, we observed an induction of STAT3 phosphorylation, and, to a much lesser extent, an induction of STAT5 phosphorylation in response to IL-23, predominantly in mucosal-associated invariant T (MAIT) and γδ T cells, and, to a lesser extent, in natural killer (NK) and memory CD4 + T cells ( Figure 2E and Figure S2H). In P5 and P6, no STAT phosphorylation was induced upon IL-23 stimulation in any of the leukocyte subsets tested ( Figure 2E and Figure S2H). By contrast, STAT phosphorylation in response to IFN-α2b was similar in cells from P5 and P6 and in the healthy controls ( Figure 2E and Figure S2H). The six patients, thus, had a complete form of AR IL-23R deficiency.

Impaired basal IFN-γ immunity ex vivo in inherited IL-23R deficiency
As a first approach to deciphering the cellular basis of MSMD and CMC in patients with inherited IL-23R deficiency, we compared the frequency of leukocyte subsets in the whole blood of healthy controls, five IL-23R-deficient patients, three IL-12Rβ1-deficient patients, and four patients heterozygous for STAT1 gain-of-function (GOF) variants (64), by CyTOF ( Figure 3A and Figure S3A), and in peripheral blood mononuclear cells (PBMCs), by spectral flow cytometry ( Figure S3B). The counts and frequencies of myeloid cells, NK cells, MAIT cells, γδ T cells, innate lymphoid cells (ILCs), total CD4 + αβ T cells, total CD8 + αβ T cells, and memory T H 1, T H 2, T H 1*, and T H 17 cells in IL-23R-deficient patients were within the range of healthy controls ( Figure 3A and Figure S3A-S3B). We then analyzed PBMC samples from P3, P4, and P5 by single-cell RNA sequencing (scRNA-seq). We simultaneously analyzed PBMCs from two IL-12Rβ1-and two IL-12Rβ2deficient patients, to investigate IL-23-dependent but IL-12-independent cellular phenotypes underlying MSMD, and from one STAT1 GOF patient, to investigate IL-23-dependent cellular phenotypes underlying CMC. Batch-corrected unsupervised clustering identified 22 different leukocyte subsets ( Figure 3B, S3C and S3D). Pseudobulk principal component analysis (PCA) revealed the transcriptional phenotypes of different leukocyte subsets of IL-23R-, IL-12Rβ1-, and IL-12Rβ2-deficient patients, the patient with a STAT1 GOF variant, and healthy adults and children ( Figure S3E). Gene set enrichment analysis (GSEA) revealed a downregulation of genes regulated by IFN-γ in most clusters identified, except for plasma cells, type 1 conventional dendritic cells and plasmacytoid dendritic cells, in IL-23R-deficient patients relative to healthy children ( Figure 3C and D). These baseline transcriptional alterations may be associated with an impairment of intercellular communication, as suggested by the probabilistic inference of intercellular communications in CellChat analysis (65) ( Figure 3E). Interestingly, GSEA identified the high mobility group box 1 (HMGB1)-regulated geneset as the only transcription factor target geneset jointly downregulated in IL-23R-deficient and STAT1 GOF T H 17 cells ( Figure 3F), suggesting that a deficiency of HMGB1 activity may underlie impaired T H 17-dependent immunity in both IL-23R-deficient and STAT1 GOF patients. Overall, these results suggest that IL-23R deficiency impairs IFN-γ immunity in at least 19 leukocyte subsets in vivo, without affecting their development per se.

IL-23 induces IFNG in NK, MAIT and Vδ2 + γδ T cells
We delineated IL-23-dependent cellular responses further by analyzing PBMCs from two IL-23R-deficient (P4 and P6) patients, one IL-12Rβ1-deficient patient, one STAT1 GOF patient, and six healthy individuals (including P4's sister), by scRNA-seq, after incubation with or without IL-23 for six hours. Clustering analysis identified 18 leukocyte subsets ( Figure 4A and S4A). GSEA identified IFN-γ-regulated genes with IL-23-driven induction in healthy controls, the expression of which was weaker in several leukocyte subsets in IL-23R-and IL-12Rβ1-deficient patients, including NK cells, Vδ2 + γδ T cells, monocytes, and myeloid dendritic cells (mDC). (Figure 4B and S4B). This observation suggested that IL-23 stimulation directly induces IFNG expression. Indeed, upon IL-23 stimulation, we observed an increase in the percentage of NK, MAIT, and Vδ2 + γδ T cells expressing IFNG in healthy controls and the STAT1 GOF patient, but not in IL-23R-or IL-12Rβ1-deficient patients ( Figure 4C). In comparisons of IL-23R-or IL-12Rβ1-deficient patients with healthy controls, IFNG was the gene for which IL-23-driven induction was most strikingly impaired in Vδ2 + γδ T, MAIT, and NK cells ( Figure 4D). By contrast, no induction of IL17A or IL17F was detected in IL-23-stimulated cells from healthy controls. Thus, human IL-23 acts predominantly as an early IFN-γ-inducing cytokine in innate-like adaptive (Vδ2 + γδ T, MAIT) and innate (NK) lymphocytes, which, in turn, induce IFN-γ-dependent transcriptional programs in both lymphoid and myeloid leukocyte subsets.

Impaired ex vivo IL-23-mediated induction of IFN-γ in inherited IL-23R deficiency
Human IFN-γ is essential for antimycobacterial immunity, as at least 36 of the 37 known genetic etiologies of MSMD affect the production of, or response to IFN-γ. Given the IL-23-dependent induction of IFNG at mRNA level observed on scRNA-seq, we performed ex vivo experiments to test the hypothesis that inherited IL-23R deficiency impairs IFNγ production in response to mycobacteria. We used spectral flow cytometry to assess intracellular IFN-γ production by lymphocyte subsets. Following IL-23 stimulation, the frequency of IFN-γ-expressing NK, MAIT, Vδ2 + γδ T, and to a lesser extent CD4 + T cells in healthy controls increased relative to unstimulated cells, whereas no induction of intracellular IFN-γ was observed in IL-23R-deficient cells ( Figure 4E). Following infection with BCG, intracellular IFN-γ induction was mildly impaired in CD4 + T cells and more severely in MAIT and Vδ2 + γδ T cells of IL-23R-deficient patients, and this impairment was not rescued by exogenous IL-12 ( Figure 4E). Intracellular IFN-γ production in response to BCG infection was not significantly impaired in the NK of the IL-23R-deficient patient ( Figure 4E). These results are consistent with the IL-23-dependent induction of IFNG expression observed in MAIT and Vδ2 + γδ T cells from healthy controls on scRNAseq. Overall, IL-23R deficiency impairs IFN-γ production upon mycobacterial infection ex vivo; this impairment involves at least MAIT and Vδ2 + γδ T cells, and leads to insufficient IFN-γ-dependent protective immunity to mycobacteria.

Normal development of BCG-specific CD4 + memory T cells in IL-23R-deficient patients
Our results suggested that IFN-γ induction by MAIT and Vδ2 + γδ T cells was deficient in IL-23R-deficient patients, but we could not rule out a contribution of impaired memory CD4 + T cell function to the mycobacterial disease in these patients. We therefore sorted naïve and memory CD4 + T cells from P4, P5, and healthy controls, and cultured them for five days under T H 0 (T-cell activation and expansion [TAE] beads only) or T H 1 (TAE beads plus IL-12) polarizing conditions. IFN-γ was induced in T H 1-stimulated naïve CD4 + T cells from the IL-23R-deficients patients, but the amounts in which it was produced and secreted (assessed by determining the percentage of IFN-γ + cells and the concentration of IFN-γ in the supernatant) were at or below the lower end of the range for naïve CD4 + T cells from healthy donors ( Figure 5A and S5A). Similar results were obtained for IL23R-deficient memory CD4 + T cells in that these cells were able to produce and secrete IFN-γ, but in smaller amounts than CD4 + T cells from healthy donors ( Figure 5B and S5B). We then assessed the impact of IL-23R-deficiency on the proportion of BCG-reactive memory CD4 + T cells and their IFN-γ production. Following stimulation with tuberculin purified protein derivative (PPD), the proportion of IFN-γ + cells among CD40L + /CD69 + reactive memory CD4 + T cells was similar between healthy controls and the IL-23R-deficient patients ( Figure S5C). Following polyclonal stimulation of T cells with antibody complexes binding CD2, CD3, and CD28, the proportion of CD40L + /CD69 + activated memory CD4 + T cells, including that of IFN-γ + cells, was lower in IL-23R-deficient patients, when compared with healthy controls ( Figure S5C). Finally, we assessed the reactivity to BCG of memory CD4 + T cells expanded in vitro with PHA, IL-2 and irradiated allogeneic PBMCs; the frequency of BCG-specific memory CD4 + T cells was similar between healthy controls and IL-23R-deficient patients ( Figure 5C and Figure S5D and S5E). Most of these cells belonged to the CCR6 + compartment ( Figure 5D and Figure S5F), suggesting that IL-23R-deficiency does not impair the development of BCG-specific T H 1* cells (66). IFN-γ production by BCG-specific memory CD4 + T cells was also similar between IL-23R-deficient patients and healthy controls ( Figure 5E). Overall, IL-23R deficiency slightly decreased the capacity of CD4 + T cells to differentiate into IFN-γ-producing cells, but did not appear to affect the development of BCG-specific memory CD4 + T cells. The normal frequency of BCGspecific CD4 + memory T cells and their intact ability to produce IFN-γ relative to healthy individuals may account for the absence of mycobacterial disease recurrence in the seven IL-23R-deficient patients described to date (11, 67) (see Table S1).

Impaired ex vivo IL-23-mediated production of IL-17 cytokines in inherited IL-23R deficiency
IL-17A/F are cytokines essential for anti-Candida mucosal immunity, as all 14 known genetic etiologies of CMC (isolated or syndromic) affect IL-17-mediated immunity (36,68). We therefore performed ex vivo experiments to assess the impact of inherited IL-23R deficiency on IL-17-dependent immunity to Candida. We first assessed the responses of PBMCs from P3, P4, and P5 to ex vivo stimulation for eight hours with phorbol 12myristate 13-acetate and ionomycin (P/I). We measured intracellular IL-17A production in different lymphocyte subsets by spectral flow cytometry. Following P/I stimulation, the frequencies of IL-17A + MAIT and CD4 + T cells were significantly lower in IL-23Rdeficient patients than in healthy controls ( Figure 6A). We assessed the response of IL-23Rdeficient PBMCs to ex vivo stimulation with IL-23 for 48 hours. We observed no increase in the frequency of IL-17A + MAIT cells upon IL-23 stimulation in IL-23R-deficient patients, contrary to our findings for healthy controls ( Figure 6A). Frequencies of IL-17A + Vδ2 + γδ T, NK, and CD4 + , cells did not increase upon IL-23 stimulation in healthy controls.
We then assessed the responses of IL-23R-deficient PBMCs to ex vivo stimulation with heat-killed C. albicans (HKCA). HKCA stimulation induced the secretion of IL-17A/F and IL-22 in healthy control PBMCs, but not in PBMCs from P3, P4, P5, and P6 ( Figure  6B and Figures S6A and B). Finally, we stimulated PBMCs from healthy individuals and IL-23R-deficient patients with IL-23, with or without IL-1β, in the presence or absence of HKCA. We found that the stimulation of control PBMCs with IL-23 in the presence of IL-1β enhanced the production of IL-17A, IL-17F and IL-22 relative to stimulation with IL-1β or IL-23 alone, and that IL-23 further induced IL-17F production in the presence of HKCA. By contrast, under the same stimulation conditions, no IL-17A, IL-17F, or IL-22 was detected in the supernatant of PBMCs from IL-23R-deficient patients ( Figure 6B and Figure S6B). Overall, MAIT cells from IL-23R-deficient patients, like those of IL-12Rβ1deficient patients or STAT1 GOF patients, displayed impaired IL-17A induction upon IL-23 stimulation, and PBMCs from IL-23R-and IL-12Rβ1-deficient patients displayed impaired IL-17A/F production in response to heat-killed C. albicans, possibly accounting for their risk of CMC (41)(42)(43)(44)(45)(46)(47).

Normal development of C. albicans-specific CD4 + memory T cells in IL-23R-deficient patients
CMC displays incomplete penetrance in IL-23R deficiency, suggesting that IL-17 production may be preserved in some cell subsets. We therefore studied the impact of IL-23R deficiency on the development of T H 17 cells. We sorted naïve and memory CD4 + T cells from P4, P5, and healthy individuals. After five days of culture under T H 17 polarizing (TAE beads plus TGF-β, IL-1β, IL-6, IL-21 and IL-23) conditions, the production of IL-17A and IL-17F (assessed by determining the percentage of IL-17A + and IL-17F + cells among naïve and memory CD4 + T cells, and the concentration of IL-17A and IL-17F secreted by naïve and memory CD4 + T cells) by IL-23R-deficient cells was impaired relative to that of healthy controls, but not entirely abolished ( Figure 5A and B, and Figure S5A and B). We then assessed the reactivity to C. albicans of in vitro-expanded CD4 + memory T cells from P2, P3, P4, P6 and healthy controls; the frequency of C. albicans-specific cells was similar for healthy controls and IL-23R-deficient patients ( Figure 5C and Figure S5D).
Most of these cells belonged to the CCR6 + compartment ( Figure 5D), suggesting that IL-23R-deficiency does not impair the development of C. albicans-specific T H 17 cells (66). Moreover, IL-17A production by C. albicans-specific CD4 + memory T cells was similar for IL-23R-deficient patients and healthy controls ( Figure 5F). Overall, these results suggest that IL-23R deficiency slightly decreases the capacity of CD4 + T cells to differentiate into T H 17-producing cells but does not impair the development of C. albicans-specific CD4 + memory T cells. The normal frequency of C. albicans-specific CD4 + memory T cells and the conservation of their capacity to produce IL-17A may be sufficient to protect against CMC, in some patients, potentially accounting for the incomplete penetrance of CMC in IL-23R-deficient patients (36,(41)(42)(43)(44).

DISCUSSION
We report the characterization of six patients from four unrelated Iranian kindreds with AR complete IL-23R deficiency. These patients have normal numbers of circulating leukocytes from various subsets, including cell types that normally express IL-23R, such as NK, iNKT, MAIT, Vδ2 + γδ T, T H 1, T H 17 and T H 1* cells (11,69). The vulnerability of some IL-12p40-, IL-12Rβ1-, and IL-23R-deficient patients to CMC suggests that human IL-23 plays a crucial role in IL-17A/F-mediated defense against mucocutaneous infections with Candida spp. in a subset of affected individuals. Indeed, CMC is seen in ~ 25 to 35% of patients with any of these three disorders, implying that IL-23 is redundant for defense against Candida in most individuals. We observed an IL-23driven induction of IL-17A ex vivo only for MAIT cells from healthy individuals, this induction being abolished in IL23R-deficient patients. We also found impaired IL-17A/F secretion by IL-23R-deficient leukocytes in response heat-killed C. albicans, possibly due to the impaired IL-23 responses of MAIT cells. In a mouse model of experimental autoimmune encephalomyelitis (EAE), IL-23 drives the expansion of the IL-17A + CD4 + Tcell population (70). IL-23-deficient mice (Il23p19 −/− ) have a higher C. albicans burden than WT mice in models of oropharyngeal candidiasis (OPC) and cutaneous C. albicans infection (71,72). The molecular and cellular basis of CMC in patients with IL-23R deficiency is probably similar to that in IL-12p40-and IL-12Rβ1-deficient patients, whose leukocytes also display no IL-23 signaling. In patients with any of these three disorders, CMC displays incomplete penetrance, which does not seem to be explained by the interindividual variability of IL-17A/F production by leukocyte subsets. Incomplete penetrance may reflect the observations that the development of C. albicans-specific memory CD4 + T cells is preserved in IL-23R-deficient patients, and that IL-17A induction is controlled by IL-23 in only one lymphocyte subset (MAIT cells), at least ex vivo.
By contrast, human IL-23/IL-23R signaling was surprisingly found to be essential for IFN-γ-mediated immunity to mycobacteria in all IL-23R-deficient patients identified. Our previous report of kindred A in 2018 was suggestive, but not conclusive, as only two siblings with MSMD were reported, and the cellular basis of the disease was not characterized (11). The data reported here, together with the recent description of disseminated environmental mycobacterial disease in an IL-23R-deficient patient (67), further suggest that human IL-23 is essential for host defense against even weakly virulent mycobacteria, as IL-23R deficiency underlies MSMD with complete penetrance in five unrelated kindreds with different and private IL23R genotypes. An ascertainment bias is unlikely to have affected this assessment, as two of the six MSMD patients are siblings of index cases. We also deciphered the cellular basis of mycobacterial disease. We showed, by scRNAseq, that inherited IL-23R deficiency impairs IFN-γ-dependent signaling in MAIT, Vδ2 + γδ T, T H 1, T H 17, and T H 1* cells in the basal state in vivo. We speculate that various environmental stimuli continuously trigger low-level IFN-γ signaling in an IL-23-dependent manner in the basal state in healthy individuals. This model is further supported by the observations that IL-23R-deficient MAIT and Vδ2 + γδ cells display impaired induction of IFNG mRNA and IFN-γ protein upon stimulation with IL-23 and live BCG mycobacteria ex vivo.
However, these findings do not exclude the possible contribution of other cellular subsets to IL-23-dependent immunity to mycobacteria, such as IL-23R-deficient CD4 + T cells, which showed decreased production of IFN-γ under some conditions. Exogenous IL-12 did not completely rescue the defective IFN-γ production of IL-23R-deficient cells, indicating that IL-12 and IL-23 control IFN-γ production in different ways. Consistently, we previously showed that a partial and selective defect of IL-23 signaling due to a common homozygous missense variant of TYK2 (P1104A) predisposes patients to TB (9,12). The physiological significance of this finding is supported by its evolutionary impact; the frequency of the P1104A TYK2 allele has decreased in Europeans over the last 2,000 years, probably due to the negative selection exerted by TB (and possibly, to a lesser extent, by other intramacrophagic pathogens) (73). In this context, our report further attests to the essential contribution of human IL-23 to antimycobacterial immunity.
Our findings therefore suggest that human IL-12 and IL-23 are functionally more closely related, via their induction of IFN-γ, than previously thought based on the classical T H 1/ T H 17 paradigm established in mice (49)(50)(51)74). The discovery of IL-23 and IL-23R in the early 2000s was associated with the definition of T H 17 cells as a distinct T-cell lineage, different from T H 1 and T H 2 cells (49)(50)(51)74). In this view, IL-12 is an IFN-γ-inducing signature cytokine, whereas IL-23 is an IL-17-inducing signature cytokine (49)(50)(51). The observation that the administration of IFN-γ -the archetypal T H 1 cytokine -protects mice from EAE, whereas Ifng deletion worsens EAE suggested that the T H 1/T H 2 dichotomy was insufficient (50,74). Subsequent findings that IL-23 promotes the expansion of a T-cell population that produces IL-17, IL-6, and TNF (70), and EAE resistance in IL-23deficient mice paved the way for the definition of T H 17 cells (75). This led to the IL-12/ IFN-γ-T H 1 and IL-23/IL-17-T H 17 dichotomy (50,51). The experiment of Nature reported here suggests that this dichotomy holds only partially in humans, and that human IL-23 is instead primarily an antimycobacterial IFN-γ-inducing cytokine. IL-12 is essential for IFN-γ production by antigen-specific CD4 + αβ T cells (66), which are purely adaptive T cells, but IL-23 is essential for IFN-γ production primarily by innate-like adaptive cells (MAIT and Vδ2 + γδ T). We, thus, speculate that IL-23 may also be the phylogenetically ancestral IFN-γ-inducing cytokine. Our findings further suggest that the mechanisms underlying the therapeutic efficacy of IL-23p19 blockers may involve an inhibition not only of excessive IL-17 immunity, if indeed this immunity is inhibited at all, but also, and perhaps predominantly or exclusively, of excessive IFN-γ immunity (51,74). They also challenge the involvement of IL-17 immunity inferred from IL-23R signals detected in genome-wide association studies (51,76,77).

Study design
We performed WES on a cohort of 802 patients with MSMD of unidentified genetic etiology. We identified six patients homozygous for one out of four different eLOF or pLOF IL23R variants. All six patients had a history of MSMD and two suffered from CMC. We examined the consequences of these mutations both in isogenic overexpression experiments and in the patients' cells. We found that the four IL23R variants abolish cell response to IL-23. We performed ex vivo and in vitro experiments using PBMCs from the patients and controls, to investigate the impact of IL-23R deficiency on IFN-γ-dependent immunity to mycobacteria and IL-17-dependent immunity to C. albicans. Conclusions were drawn from analyzing the results from aforementioned approaches collectively.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. Institutes of Health (NIH) Tetramer Core Facility (NTCF) for providing the 5-OP-RU-loaded MR1 tetramer, which was developed jointly with Dr. James McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie. The following reagent was obtained through BEI Resources, NIAID, NIH: Mycobacterium tuberculosis, strain H37Rv, whole-cell lysate, NR-14822.

Funding
The

Availability of data and materials
All raw and processed data and biological materials, including immortalized cell lines from patients, are available upon request from the corresponding authors under a material/data transfer agreement.